1. Field of the Invention
The present invention relates generally to devices and methods for optically analyzing biological fluids. In particular, it relates to the design and use of centrifugal rotors which allow peripheral cuvettes to be filled, without creation of air bubbles, as the rotor spins and the subsequent rapid optical analysis of the fluid in each cuvette.
Blood plasma and other biological fluids or material frequently require that liquids be quickly divided into a plurality of discrete aliquots so that a variety of optical tests or assays may be performed. It is also frequently desirable to first separate potentially-interfering cellular components of the material from the biological fluid prior to testing. Such separation and division steps have heretofore been typically performed by centrifugation to separate, for instance, blood plasma from the cellular components, followed by manual or automated pipetting of the blood plasma into separate test wells. Such procedures are labor intensive and time-consuming, and various automated systems and methods have been proposed for providing multiple aliquots of plasma suitable for testing in a more efficient manner.
Of particular interest to the present invention are centrifugal rotors which have been modified both to separate plasma from whole blood and to then distribute the separated plasma into separate test wells which are suitable for optically analyzing their contents. The use of such rotors can provide a plurality of discrete plasma volumes, and/or diluted plasma volumes which may be tested or evaluated, all present within the centrifugal rotor, greatly enhancing the efficiency of automated testing procedures.
Although a significant improvement over prior manual or partly manual procedures, previous modified centrifugal rotors have suffered from a number of deficiencies. Such rotors have frequently required the application of relatively large volumes of whole blood or other biological material in order to achieve the desired separation and distribution. The rotors are also not well designed for optical analysis of the contents of the individual test wells. Those rotors that allow optical analysis in situ require that the light beam pass vertically through the test well. Thus, variations in the amount of fluid in each well leads to variation in the length of the light path and can lead to variable results in the assays. In addition, without reliable methods for evacuating gas from the test wells, air bubbles introduced into the wells during filling can also affect analysis. Such air bubbles are particularly disadvantageous when optical analysis is performed using light beams passing vertically through the rotor.
Moreover, prior art rotors have frequently utilized complex designs which are difficult and costly to manufacture. Often, the rotors require various separable parts or components which are brought together or separated at different points in the centrifugation procedure. Previous centrifugal rotors have often been limited in the number of discrete samples and test wells which they can provide, and in some cases require the use of a separate displacement fluid to effect flow of blood and plasma through the system.
For these reasons, it would be desirable to provide improved centrifugal rotors and methods suitable for separating biological materials into fluid and cellular components, diluting the fluid, if necessary, and further distributing the fluid into a plurality of discrete cuvettes within the rotors. The rotors should be capable of separating and distributing relatively small volumes of fluid and should not require the use of a displacement fluid for effecting such separation. Moreover, to decrease variability between assays, the cuvettes should provide a uniform light path and should not contain air bubbles after filling.
The rotors should be able to accommodate relatively large numbers of test wells, and the rotor design should be simple and amenable to low-cost manufacturing procedures. In particular, it would be desirable if the rotors were of unitary construction with no separable or movable parts. Plasma separation methods should be simple and be capable of being performed in relatively short times. In particular, the methods should require relatively few steps and should be capable of being performed with little or no intervention or manipulations by the operator. It would be particularly desirable if the methods required only rotation of the rotor in order to effect distribution of the fluid into the cuvettes, followed by optical analysis in situ.
2. Description of the Background Art
U.S. Pat. No. 4,623,519 describes a centrifugal rotor which comprises a liquid feed and gas evacuating duct which depends on the rough surfaces of the duct to create random patterns by which fluid enters the test well and gas escapes. The liquid feed and gas evacuating flow paths are not discrete U.S. Pat. Nos. 4,154,793 and 4,762,683 relate to centrifugal rotors having peripheral cells, each cell having a separate air escape orifice to allow air to escape as the cell is filled with liquid. U.S. Pat. No. 4,412,973 discloses a centrifugal rotor having peripheral cells which are fed by two separate paths for conveying liquid to the cell. One path being for the sample, the other for reagents. U.S. Pat. Nos. 4,469,793 and 4,894,204 relate to centrifugal rotors which dispense a predetermined dose of a sample liquid into a peripheral cell on the rotor.
U.S. Pat. No. 4,652,137 relates to a centrifugal rotor comprising an array of peripheral cuvettes, the contents of which are optically analyzed using a vertical light source and detector. U.S. Pat. No. 4,695,164 describes a position detector for detecting a rotational position of a rotor of a centrifugal analyzer wherein the light beam is deflected by mirrors that are not on the rotor. U.S. Pat. No. 4,226,537 relates to an analytical centrifuge having mirrors which deflect a light beam. The mirrors are not on the rotor. U.S. Pat. Nos. 4,785,407 and Reissue No. 31,149 relate to carousel analyzers, the cuvettes of which are optically analyzed. Reissue U.S. Pat. No. 31,149 discloses the use of mirrors to deflect the light beam, the mirrors are not a part of the rotating carousel. U.S. Pat. No. 4,431,307 relates to a set of cuvettes which are adapted to optical analysis using a vertical light source and detector. U.S. Pat. No. 4,776,697 relates to an optical particle analyzer. U.S. Pat. No. 4,350,283 relates to a centrifuge rotor adapted for continuous separation of particles.